Gene therapy is an attractive and novel approach to prevent and treat diseases of the eye. However, current vectors being developed for ocular gee therapy have serious drawbacks sharply limiting their clinical usefulness. Perhaps the major problem hindering gene therapy is the inefficiency of gene transfer to slowly and non-dividing cells. Nowhere are these cells more prevalent and important than in the eye. While focus has been on developing vectors that target genes to and into cells, the fate of the DNA once inside the cytoplasm has not been addressed. Once vectors have been targeted to and entered individual cells, their DNA, whether as plasmid, viral genome o reverse-transcribed DNA, must become nuclear to function. The PI has shown that nuclear import of plasmid DNA occurs in primary cultures of human corneal epithelial cells and keratocytes in the absence of cell division. Moreover, plasmid nuclear import is sequence-specific and requires a 366 bp fragment of DNA that contains a promoter and enhancer and binding sites for several transcription factors that are abundantly expressed in the eye. The PI hypothesizes that transcription factors bind to specific DNA sequences, thereby allowing the DNA to utilize the protein import machinery for nuclear entry. It was also found that this DNA sequence increased nuclear targeting of plasmids and greatly increased gene expression in liposome-transfected cells, suggesting that these sequences will have a great impact in gene transfer in vivo. Based on these results, he hypothesizes that nuclear targeting DNA sequences will increase the efficiency of gene transfer and expression in the cornea. He will test this hypothesis in cultured human corneal cells and extend the findings to an in vivo model for herpes stromal keratitis. The specific aims are to (1) optimize specific DNA sequences needed for plasmid DNA nuclear import in human corneal cells using microinjection and in situ hybridization, and transfection and gene expression studies. (2)Optimize non-viral gene transfer to the cornea using in vivo electroporation and determine whether nuclear targeting DNA sequences improve transfer of reporter genes in vivo in the mouse eye. (3)Test the efficacy of nuclear-targeting IL-10 expressing constructs in the treatment of herpes stromal keratitis, since IL-10 is a potent inhibitor of HSV-induced necrotizing stromal keratitis. The results obtained from these studies will lead directly to the deveopment of ocular gene therapy vectors with improved transfer efficiency.